l ’ ttv ATHES! U) -~_v L 3 8 MR! uranium State University This is to certify that the dissertation entitled Altered Adrenocortical Metabolism in Vitamin B Deficient Rats 6 presented by Sue Marie Ford has been accepted towards fulfillment of the requirements for Ph.D. degree in Human Nutrition I (jbnauz ~J1 §59nal Major professor Jenny T. Date February 13, 1985 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES W RETURNING MATERIALS: Place in book drop to remove this checkout from your record. flfl§§_will be charged if book is returned after the date stamped below. ALTERED ADRENOCORTICAL METABOLISM IN VITAMIN B6 DEFICIENT RATS BY Sue Marie Ford A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1985 ABSTRACT ALTERED ADRENOCORTICAL METABOLISM IN VITAMIN B6 DEFICIENT RATS By Sue Marie Ford Previous investigations suggest that a defect in adrenocortical metabolism exists in vitamin 36-deficient rats. The symptoms are consistent with decreased glucocorticoid production, with increased mineralocorticoids. This study was undertaken to test the hypothesis that lip-hydroxylation is decreased in the adrenal glands of vitamin 36-deficient rats. TWO strains of rats which are known to differ in susceptibility to B -deficiency were used. 6 Sprague-Dawley (CD) or Wistar (WIS) male rats were fed a semi-purified diet lacking vitamin B6 or one with pyridoxine (30 mg/kg). At the end of 3 or 10 weeks the in zi££2_steroidogenic capacity of the adrenal glands was assayed. The capsule and inner cortex of the adrenals were incubated separately in Krebs-Ringer bicarbonate buffer containing glucose (200 mg/dl) and progesterone (51 uM). The steroids released into the medium were determined by high-pressure liquid chromatography. 11-Deoxycorticosterone (DOC) production from progesterone was increased in tissue from the inner zones of CD and WIS rats consuming the vitamin 36-deficient diet for 10 weeks, but not 3 weeks. The Sue Marie Ford synthesis of corticosterone and 18-OH—DOC by the inner zones of WIS deficient rats was decreased only at 3 weeks. DOC production in the capsules from deficient animals was not different than that of control. Capsular corticosterone and 18-OH—DOC syntheses were decreased at 3 weeks in deficient WIS rats, but there was no effect of the deficient diet in tissue from CD rats After 10 weeks, corticosterone synthesis was decreased in the capsules from both strains, but 18-OH-DOC production was not. Aldosterone production was markedly depressed in the capsules from both strains at 3 and 10 weeks. These results demonstrate that 11p- and 18-hydroxylases are decreased in vitamin B6—deficient rats, resulting in enhanced mineralocorticoid production (DOC) but decreased glucocorticoid (corticosterone). The effect on WIS rats is greater than CD rats. ACKNOWLEDGEMENTS I would like to thank the members of my committee, Dr. Jenny Bond, Dr. Jerry Hook, Dr. David Rovner, Dr. Rachel Schemmel, and Dr. Maija Zile for their advice and critical evaluation of this work. The technical assistance of Katie O’Toole, Kathy Boyer, Rose Heikken, Liz Bailie, Dierdre Hansen, and Jon Gieche was appreciated. The personal support of my friends, Mark Messina, Ginny Messina, Janny Seto, Mal Collum, Holly Ernst, and Lisa Lepper was invaluable. Special thanks are due to my family for their love and encouragement during my graduate studies. The financial support provided by Dr. Bond, the Department of Food Science and Human Nutrition, the College of Human Ecology, Michigan Heart Association, and the Center for Environmental Toxicology is gratefully acknowledged. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ii LIST OF TABLES - iv LIST OF FIGURES v INTRODUCTION ——— --- 1 Vitamin B 1 Discovery and Isolation l Pharmacokinetics 2 Biochemical Roles of B6 Vitamers 7 Deficiency Symptoms 14 Adrenal Glands 22 Corticosteroid Synthesis and Metabolism 22 Biological Effects of Corticosteroids 32 Effects of Vitamin B6 Deficiency on the Adrenals ---------- 36 Rationale —— 41 METHODS 45 Animals and Diets 45 Adrenal Steroidogenesis 45 Sample Extraction 49 Chromatography 51 Statistical Analyses —— 53 RESULTS 54 Body Weight 54 Organ Weights 54 Protein Content of the Adrenal Glands 59 Adrenal Steroidogenesis 59 DISCUSSION —— 83 Speculations 93 BIBLIOGRAPHY 101 iii LIST OF TABLES Table Page 1 Composition of Rat Diet 46 2 Vitamin Mix for B6 Study 47 3 Mineral Mixes for B6 Study 48 4 Effect of Vitamin B6 Deficiency on Body Weight (3 week study) 55 5 Effect of Vitamin B6 Deficiency on Body Weight (10 week study) -- 56 6 Effects of Consuming a Vitamin B Deficient Diet for 3 Weeks on Organ Weights of Rats — 57 7 Effect of Consuming a Vitamin B Deficient Diet for 10 Weeks on Organ Weights of Ra s 58 8 Protein Content of Adrenal Glands 6O 9 Summary of Data From Figures 4 - 13 97 iv Figure 10 ll 12 13 LIST OF FIGURES Pathways of metabolism and interconversion of B Vitamers 6 Formation of a Schiff base between pyridoxal (PL) or pyridoxal phosphate and an amino acid Pathways of steroidogenesis in the adrenal cortex -— ----- Effect of diet and strain on formation of steroids from progesterone by quartered decapsulated (inner) adrenal glands Effect of diet and strain on formation of steroids from progesterone by capsular portion of adrenal glands ----- Effect of diet and strain on formation of ll-deoxy- corticosterone (DOC) from progesterone by quartered decapsulated (inner) adrenal glands Effect of diet and strain on formation of 11-deoxy- corticosterone (DOC) from progesterone by capsular portion of adrenal glands Effect of diet and strain on formation of cortico- sterone (B) from progesterone by quartered decapsulated (inner) adrenal glands Effect of diet and strain on formation of cortico- sterone (B) from progesterone by capsular portion of adrenal glands Effect of diet and strain on formation of 18-hydroxy- ll-deoxycorticosterone (18-OH—DOC) from progesterone by quartered decapsulated (inner) adrenal glands --—----- Effect of diet and strain on formation of l8-hydroxy- ll-deoxycorticosterone (lB-OH-DOC) from progesterone by capsular portion of adrenal glands Effect of diet and strain on formation of aldosterone from progesterone by quartered decapsulated (inner) adrenal glands Effect of diet and strain on formation of aldosterone from progesterone by capsular portion of adrenal glands -- Page 25 62 64 67 69 71 73 75 77 79 INTRODUCTION Pyridoxine was isolated and identified as a required nutrient for rats in the 19303 and 40s. Among the earliest symptoms resulting from deficiency of this vitamin include histological changes in the adrenal gland indicative of altered function. During this same period of time, comprehensive studies of the adrenal gland were undertaken. Although numerous investigators studied the relationship between vitamin B6 deficiency and the adrenals, knowledge of the structure and functions of the steroids produced by the cortex was inadequate, as was the methodology available to study such a problem. Since then, the major corticosteroids in the rat have been isolated and identified and more is known about the control of adrenal steroidogenesis. The recent increase in availability of high pressure liquid chromatography makes practical the separation and quantification of such compounds produced by the adrenal glands of the rat. The objective of this work will be to review the literature regarding this problem, and test the hypothesis that steroid synthesis is abnormal in the adrenal glands of vitamin B deficient rats, specifically that the activity of 11 6 -hydroxylase is depressed. VITAMIN 36 Discovery and Isolation The isolation and identification of the individual components of the B-complex vitamins in the early part of this century represents one of the most exciting periods in nutrition research. The initial impetus to such work was the recognition that the human diseases 2 beri-beri and pellegra were due to dietary inadequacies rather than infectious agents or toxicities. Numerous laboratories around the world became engaged in intense competition to discover the specific nutrients. The isolation and purification of vitamin B was 6 accomplished in 1938 by four independent labs (Lepkovsky, 1979). The observation that the various test organisms had different growth responses to the various forms of the vitamin B (Snell & 6 Rannefeld, 1945) led to the development of assays for the individual compounds in natural materials. The fact that they were interchangeable in animal nutrition (Snell & Rannefeld, 1945) provided the first clues regarding the metabolism of vitamin B6. Pharmacokinetics The structures and official nomenclature of the vitamin B6 compounds are shown in Figure 1. "Vitamin B6" is the recommended term for all of the substances derived from 3-hydroxy-2-methy1pyridine which "exhibit the biological activity of pyridoxine in rats" (Mayes et al., 1974). In animals each of the B6 vitamers is readily converted to the biologically active forms and thus are equally potent in supporting growth. Figure 1 shows the known pathways of metabolism and interconversion, as well as the oxidation to the biologically inactive metabolite 4’-pyridoxic acid (PA). The numbers refer to the enzymes which catalyze the designated reaction(s). The unphosphorylated forms are more commonly found in foods. They are absorbed in the intestine by passive diffusion, as shown by kinetic analyses of transport behavior (Yamada & Tsuji, 1980). Although there is some intracellular accumulation evident at low doses, this is .Aoon .Hfiannmz a somehooozv Aan amaammoueznae ovaaaeaa no Asev ommvfixo ovmsouam an wonmamumo ma A onu mo coaum>wuomca one .Ao* .mav mommcfiawwcmuu mo mcofiuom mam mp mauom ovmsovam ecu Cu wouum>noo an omHm sea mzm was an .A-a~ .Hcanesfloo a Aoouzv mm was Amqmv oumnamona meowfiuha .movmnonam onu ou mauom Hosooam was ocaam onu mo nofiumwwxo ecu mouhamumo Sofia: mahuao owaomouho n ma Asev ommvaxo AmZmV oumnmmofia o:HmeovHuhm .Amzmv mumsamosm ocaxowfiuhm oNHHHu: uoc Haas usn .Am cu zm mo defimum>cou onu mommamumo Amev owmpaxo mcfixOpHumm .Aome .wq w mamasqv mmmumsamosa oaHmeHm wcfivsaucfi .mmmmumzmmona m30fium> m hp vamaHmEooum mu AmaV coauaaauonamosama .Aokoa .Hamxmam a Hamamv Azmv ocaaaxoeansa mam .Azmv oconvHuma .Aamv meovfiuza moumamuosawoza coax: messes afiaomOuho n ma Aaav mmmcax meowaumm .Amvcoquomou woumcwfimov onu muuamumu SUHSB moehucm ecu ou mommy wuonasa one .muoEmuH> om mo cowmum>coououcw can EmHHonmuoE mo m%m3:umm .H ouswfim 2...: 9% unoccEm uv / o I Al _ Iowxo \ o: 0 ® Iowxo \ 1000 ~ mmsmwm «2Q» m2\XOQ\¢xQ z in: _ x c Ioaxu Iowxo mu «4%» 4- I, 10 with amino or keto acids (Walsh, 1979). Furthermore, the nitrogen at position 1 can be reversibly protonated, allowing for the flow of electrons in and out of the ring (Barker, 1971). Following the formation of the Schiff base with the amino group of an amino acid, a number of reactions are possible (Figure 2). Under appropriate conditions the B6 vitamers can perform the same reactions in_zi££2 as in_vizg_; however a mixture of products results (Barker, 1971; Walsh, 1979). In contrast, when associated with an enzyme the reaction is directed toward the formation of specific product(s) (Dunathan, 1971; Walsh, 1979). Transaminases are ubiquitous in mammalian tissues and catalyze transfer of the amino group from an amino acid to a receptor Gi-keto acid. These enzymes are necessary for utilization of glucogenic amino acids and the synthesis of non-essential amino acids. Transamination occurs as a two-step process (Walsh, 1979). PLP bound to the enzyme forms a Schiff base with the substrate amino acid and bond a (Figure 2) is broken. The complex is thus resolved into PMP—enzyme and the product keto acid. A second Schiff base is formed with the PMP and the recipient keto acid. When this bond is broken, PLP is regenerated and the new amino acid released. Decarboxylation involves cleavage of bond b, with release of C02 and an amine. Many biologically active amines are formed by the action of PLP-dependent decarboxylases (Holtz & Palm, 1964). Glutamic acid decarboxylase catalyzes the synthesis of gamma-aminobutyric acid (GABA), and histamine is formed by histidine decarboxylase. Aromatic amino acid decarboxylase will form the neurotransmittor dopamine from the precursor dihydroxyphenylalanine 11 (DOPA), and serotonin is derived from the decarboxylation of 5-hydroxytrypt0phan. Additionally, synthesis of the heme precursor 5-aminolevulonic acid (ALA) is catalyzed by ALA synthetase, a decarboxylase. Serine hydroxymethylase (serine aldolase) is an unusual enzyme which requires both PLP and an additional cofactor, tetrahydrofolate (THF). This enzyme catalyzes the C -C cleavage of serine to glycine with the ultimate formation of N5, Nlo-methylene THF, a compound important in the synthesis of purines and pyrimidines (Walsh, 1979). Kyneurinase, an enzyme involved in tryptophan metabolism, is an example of‘fi -replacement. Cystathionase is found in the pathway of methionine metabolism, and represents d(-elimination. Numerous xenobiotics and therapeutic agents interfere with the biological actions of vitamin B A chemical may form a stable 6' Schiff base with the PLP molecule, thus making the coenzyme unavailable for association with an enzyme and/or reaction with the substrate. Furthermore, the urinary excretion of this complex may result in depletion of PLP from the body. Penicillamine is thought to act by this mechanism (DeJesus Sevigny et al., 1966). Some compounds are inactive analogs of the coenzymatic forms of vitamin B They can 6. associate with B ~requiring enzymes, but are unable to support the 6 catalytic action of the enzyme. The classic B6 antagonist, 4’-deoxypyridoxine (DOP) may be phosphorylated by PL kinase and subsequently compete with PLP for enzymatic binding sites (Umbreit & Waddell, 1949). The action of enzymes involved in the interconversion of the vitamers may be affected. For example, PL kinase may be inhibited by isonicotinic acid hydrazide and other PL analogs inhibit 12 PL kinase, preventing formation of the phosphorylated coenzymes (Cornish, 1969; Mizuno et al., 1980) Phosphorylated derivatives of vitamin B6 also inhibit PNP oxidase (Snell & Haskell, 1970). Many antimetabolites have significant physiological actions, and have often been used to hasten the development of experimental vitamin B6 deficiency in animals and humans. However, these compounds may have effects other than on B6 metabolism. Penicillamine is an effective chelator of several trace elements, and deleterious effects on copper status may be more significant than effects on B6 nutriture (Heddle et al., 1963; Takeda et al., 1980). The rate of depletion of PLP from several tissues with isoniazid treatment (DeJesus Sevigny et al., 1966) or DOP (Stoerk, 1950a) may not duplicate that of vitamin depletion alone. Although antimetabolites may be useful for certain studies, the syndromes produced by administration of these agents may not be valid models of vitamin B deficiency 6 (Coburn & Mahuren, 1976). The activity of most vitamin B6-dependent enzymes is responsive to the level of B6 nutriture, as well as other dietary and hormonal factors. Several mechanisms are possible to explain the loss of activity which occurs during vitamin deficiency. Enzymes which bind the coenzyme loosely or those which contain subunits that depend on PLP for association may lose PLP during depletion. In such cases, the enzyme may be reactivated by adding exogenous PLP EEHXAEEE (Greengard, 1964). In contrast, the activity of alanine aminotransferase was lower in deficient animals but could not be stimulated by PLP (Lee et al., 1977). This was determined to be due to synthesis of defective apoenzyme in the absence of the coenzyme. 13 The concentration of the apoenzyme may change as the result of vitamin B6 deficiency. The rate of degradation of several B6-dependent enzymes appears to be correlated with the affinity for the coenzyme and it has been suggested that for such enzymes the coenzyme protects the apoenzyme against degradation (Anonymous, 1978). However, Lee et al. (1977) have argued that the degradation rate and the affinity for coenzyme reflect a common structural feature of the protein rather than being causally related. Katunuma (1973) has described proteases which degrade several PLP-dependent enzymes and act by rendering the enzymes susceptible to attack by the non-specific proteolytic enzymes in the cell. The presence of the coenzyme will protect the protein against degradation by the PLP enzyme degrading proteases. These proteases do not degrade hepatic tyrosine aminotransferase (Katunuma, 1973); however, inactivation of this enzyme occurs in homogenates from the liver of vitamin B6-replete rats (Reynolds 8 Thompson, 1974), but not from similar preparations from deficient rats (Reynolds, 1978; Sloger et al., 1978). This may explain the observation of Hunter and Harper (1977) that basal and glucocorticoid-induced tyrosine aminotransferase (TAT) was higher in deficient animals. The synthesis and degradation of vitamin B6 dependent enzymes such as TAT may be controlled by separate mechanisms which are individually responsive to dietary and hormonal factors. Thus, in order to examine the regulation of enzymatic activity, studies must be conducted in which dietary (vitamin deficient vs. replete) and hormonal factors (adrenalectomized, sham-Operated) are considered in concert. Recent evidence suggests that PLP may have a role in the actions 14 of steroid hormones in the cell. Cidlowski and Thanassi (1981) have proposed that PLP facilitates the recycling of steroid receptors. PLP would both increase the activation of the steroid/receptor complex in the cytosol (Sekula et al., 1982) as well as hasten its dissociation from the nuclear DNA (Cake et al., 1978). In this manner the availability of the receptor for the next steroid molecule would be increased. Such actions of PLP on steroid receptors have been observed with the glucocorticoid receptor (DiSorbo et al., 1980), the prostate androgen receptor (Hipakka & Liao, 1980; Mulder et al., 1980), and the uterine progesterone receptor (Chen et al., 1981). Deficiency Symptoms Within two to three weeks after the introduction of a vitamin B6 deficient diet, the food intake of weanling rats decreases compared to the intake of rats fed a control diet. The deficient animals grow at a slower rate and the body weight reaches a plateau, usually after 6-7 weeks. The weight gain per 100 g food ingested is less in vitamin B6 deficient rats than that of animals pair-fed a control diet (Sure & Easterling, 1949). The first symptom by which vitamin B deficiency was 6 distinguished from that of other B complex vitamins was a form of dermatitis termed acrodynia. The skin in the affected areas is scaly, often with a bloody exudate. In rats, the lesion involves the snout, paws, ears, and tail. In extreme cases, the tail curls rigidly into a flat spiral which does not straighten out under anesthesia (Stoerk, 19508). MicroscOpically the tissues exhibit hyperkeratinosis, intercellular edema, congested capillaries, atrophy of hair follicles 15 and sebaceous glands, and infiltration with inflammatory cells (Antopol & Unna, 1942). Although alopecia has been noted in some cases, the condition does not become severe. Humans also develop dermatitis around the mouth and eyes during experimental B deficiency (Vilter 6 et al., 1953). Vitamin B6 has several important roles in the functioning of the nervous system. Pyridoxal phosphate is a coenzyme for enzymes which catalyze the synthesis of biogenic amines such as serotonin, GABA, catecholamines, dopamine, and taurine (Holtz & Palm, 1964; Ebadi, 1978). Convulsions occur in deficient animals (L00, 1980) and were observed in human infants fed a vitamin B6 deficient formula (Coursin, 1955). The synthesis of sphingomyelin requires vitamin B6 (L00, 1980) and the lipid composition of the brain is altered during deficiency of the vitamin (L00, 1980). Depletion of B6 resulted in decreased synthesis of cholesterol, cerebrosides, long chain fatty acids, and myelin (L00, 1980). Peripheral neuritis has been reported in adult humans consuming a vitamin B6 deficient diet plus an antimetabolite (Vilter, et al., 1953). Vitamin B deficiency has deleterious effects on the immune 6 system. The lymphatic tissues of deficient monkeys and rats atrophy, with decreased density of small lymphocytes in the thymus and an increase in the number of abnormal cells (Robson & Schwarz, 1980). Impaired cellular immunity is also manifested as the inability of lymph cells in yitrp to respond to genetically dissimilar cells (Robson & Schwarz, 1980). The effects of B6 deficiency on the immune system are not mediated by the adrenal cortex or inanition (Agnew & Cook, 1949), but are likely to be due to impaired synthesis of nucleic acids 16 (Robson & Schwartz, 1980). Pyridoxal phosphate is involved at several points in the metabolism of tryptophan (Henderson & Hulse, 1978). The conversion of tryptophan (TRP) to tryptamine or 5-hydroxytryptophan to serotonin require PLP-dependent decarboxylases. The most quantitatively important pathway, however, is that from TRP to xanthurenic acid or N’-methylnicotinamide. Following administration of a tryptophan load, a larger amount of kynurenine is formed from TRP. Kynurenine accumulates in the cytosol and migrates into the mitochondria. In the mitocondria, kynurenine is hydroxylated, because the affinity of the hydroxylase for kynurenine is 100 times that of the transaminase. Further metabolism of 3-hydroxykynurenine by PLP-dependent enzymes in the cytosol is depressed in the vitamin deficient state, whereas that in the mitochondria is affected to a lesser degree, resulting in preferential production of xanthurenic acid. Vitamin B6 also participates in the metabolism of sulfur-containing amino acids (Sturman, 1978). Increasing the amount of methionine has been noted to aggravate the growth depressing effects of a vitamin B6 deficient diet (DeBey et al., 1952). PLP-requiring enzymes occur in the biosynthetic pathway for taurine, which is required for growth and functioning of the brain. In addition, the synthesis of the polyamines putrescine, spermidine, and spermine require vitamin B6 in ornithine decarboxylase; the role of PLP in S-adenosyl methionine decarboxylase is not certain (Pegg, 1977). Vitamin B is involved in several pathways of amino acid 6 metabolism, and the effects of deprivation on intermediary metabolism have been studied extensively. The symptoms of vitamin B6 17 deficiency are aggravated when a high-protein diet is used, and the requirement for the vitamin appears to increase with the protein content of the diet (Canham et al., 1969; Holtz & Palm, 1964). However, Williams (1964) observed that increased pyridoxine improves growth when rats are fed low protein diets which are also limited in an amino acid. It has been suggested that the observed effects of B6 deficiency on protein synthesis, as measured by amino acid incorporation, represent alterations in amino acid precursor pools (Okada & Suzuki, 1974). Deficiency of vitamin B6 alters the utilization and the cellular uptake of amino acids which would affect availibility (Anon., 1979). The net effect of vitamin B6 deficiency on protein metabolism would depend on several factors, which include: changes in enzyme activities accompanying increased protein and/or decreased B intake, the amino acid composition of the 6 protein, the rates of individual B -dependent reactions, and 6 endocrinological sequelae of changes in protein and B6 nutriture (Williams, 1964). A role of vitamin B6 in lipid metabolism has long been suspected; however, reports in the literature are conflicting and inconclusive. A decrease in the proportion of arachadonic acid in the lipids from tissues of deficient rats has been reported (Mueller, 1964). Arteriosclerotic lesions were observed in the arteries of monkeys (Greenberg, 1964). Studies on cholesterol metabolism have proven to be equally contradictory, with reports of hypercholesteremia, no change, and hypocholesteremia in deficient animals (Mueller, 1964). It is difficult to compare the results of 18 studies, due to differences in the fat and protein composition of the experimental diets (Mueller, 1964). Sabo and coworkers (1971) concluded that although B6 deficient animals have less body fat, the ability of liver and adipose tissue to synthesize lipid may be greater than that of controls if glucose is available. They also noted that insulin administration increased the activity of glucose-6-phosphate dehydrogenase to a larger extent in deficient animals (502) than in similarly treated contols (13%). Further studies by this group indicate that changes in lipid and glucose metabolism in adipose tissue from deficient rats may be secondary to increased transport of glucose into the cells ig_31££g_ (Ribaya & Gershoff, 1977). Many of the effects of B6 deficiency on intermediary metabolism have been ascribed to actions on endocrine systems. Insulin administration can prevent several of the symptoms of B6 deficiency, including changes in fatty acid composition (Gershoff, 1968), decreased appetite and impaired lipid storage (Beaton et al., 1956). Insulin activity was found to be reduced in the plasma and pancreatic tissue from deficient rats. Additionally, Singh (1980) observed diminished amylase content in acinar cells of the pancreas, as well as impaired nucleic acid and protein metabolism. It is possible that the effects on insulin production result from similar depression of pancreatic metabolism. Early studies indicated that pituitary function is affected in vitamin B6 deficiency. Administration of growth hormone (GH) to deficient animals resulted in a growth response which was not observed in control animals (Huber & Gershoff, 1965). Deficient rats had low 19 pituitary and serum GH levels compared to ad libitum (Huber & Gershoff, 1965) but not pair-fed controls (Rose, 1978). The vitamin 86 content of the pituitary was found to be high compared to other organs (Huber & Gershoff, 1965), suggesting a possible role of the vitamin in the functioning of the gland. Beare and coworkers (1953a) observed that administration of GH aggravated the effects of the deficiency (severity of acrodynia, increased BUN, decreased thymus weight, increased adrenal weight). Administration of the GH to pyridoxine-sufficient rats also increased the adrenal weight and decreased thymus weight. The follicle-stimulating activity (FSH) in the pituitary of vitamin B6-deficient rats was greatly increased compared to controls, whereas interstitial cell stimulating hormone (ICSH; lutenizing hormone) and prolactin were only slightly affected (Wooten et al., 1955). However, the amount of FSH necessary to stimulate follicular growth was much higher in deficient rats (Wooten, 1958). The authors suggested that the release of FSH was impaired in vitamin B6 deficient rats. Increased kidney weight and kidney/body weight ratios have been reported in vitamin B6-deficient rats (Olsen & Martindale, 1954; Agnew, 1955). Agnew (1949, 1951) observed hematuria in hooded Lister rats maintained on a B6 deficient-diet for longer than 11 weeks. Microscopic examination of the kidneys from these animals revealed fibrosis and areas of calcification at a much greater frequency than found in rats fed a control diet. Albino (Wistar) rats did not develop hematuria or fibrosis, thereby demonstrating a marked strain difference in response to the deficient diet. Lesions were observed in the kidneys of rats fed a vitamin B6 deficient diet for 33 weeks 20 (Reynolds & Slaughter, 1980). These consisted of focal necrosis, inflammation, and regenerative hyperplasia in the tubules; however, data were not available comparing the various nephron segments. Elevated levels of protein in the urine of deficient male rats were reported by Linkswiler and coworkers (1952). The proteinuria was aggravated by increasing the percentage of protein in the diet, and still further by increasing the amount of methionine. This was not a non-specific result of a dietary deficiency inasmuch as rats fed a low-protein diet exhibited no change in amount of protein excreted, and pantothenic acid-deficient rats excreted less protein. Davis and Sloop (1965) reported decreased renal concentrating ability in rats maintained on a vitamin B6-deficient diet for five months. This defect was apparent only when both the deficient rats and pair—fed controls were given urea in the drinking water, a manipulation which increased the maximum osmolality of the urine in both groups. No histological abnormalities were found in any area of the kidney in this study. Blood urea nitrogen (BUN) in vitamin B6 deficient animals has been reported by several groups to be increased (Lyon et al., 1958; DiPaolo et al., 1974; Hawkins et al., 1946). The increase was found to be strain- and age- dependent for mice (Lyon et al., 1958). In young mice, which are usually more susceptible to B deficiency than 6 adults, no consistent difference in BUN could be found during six weeks on the experimental diets. Furthermore the rise in BUN of adult animals was transient, and exogenously administered urea was cleared from the blood as rapidly as in animals with no increase in BUN (Beaton et al., 1953). 21 Olsen and Martindale (1954) reported that the sodium space of the B deficient rats was elevated (26.4Z.i,0.8) but not 6 significantly different from control animals (23.8Z.i.0.8). Hsu and Kawin (1962) demonstrated that plasma volume was higher in deficient rats (3.24.i..08 ml/100 gm BW), though again not significantly different from controls (2.80:: .04 ml/100 gm BW). Hsu et a1. (1958) postulated that the elevated serum sodium concentration in B6 deficient rats which they observed was responsible for the hypertension noted by others. Based on their observation of enhanced KCl preference in deficient rats, Stewart and Bhagavan (1982) further suggested that B deficiency is associated with hyperaldosteronism. 6 An association between deficiency of B-complex vitamins and increased blood pressure was first noted in 1942 by Calder. Subsequent studies indicated that vitamin B6deficiency in rats produces an elevation in blood pressure. Olsen and Martindale (1952) noted a rise in systolic pressure which reached a maximum (15 mm Hg) after four months on the deficient diet. Inclusion of DOP in the diet elicited no further increase, and supplementation of the diet with PL resulted in a rapid increase in weight and a concomitant fall (20 mm Hg) in blood pressure. Further studies showed that dietary DOP could produce increases in the blood pressure of control rats (Olsen & Martindale, 1954). The blood pressure of rats was found to increase during the first seven weeks on a vitamin B6 deficient diet, but decreased slightly between the 7th and 14th weeks (DeLorme et al., 1975). Plasma renin acitvity and angiotensin concentration in vitamin B -deprived 6 animals were twice that of the controls. The measurements were taken 22 in the eighth week of the study, several weeks after the blood pressure of the deficient animals had reached a plateau. At this time the animals were significantly affected by the vitamin depletion; the authors noted cessation of growth with the appearance of acrodynia by five to six weeks. At an advanced stage of deficiency it is possible that the effects on renin activity and angiotensin II could be the result of a debilitated state; for example, Brown and Pike (1960) reported hypOproteinemia in vitamin B6 deficient rats. It would be difficult to conclude from the data presented that the increased plasma renin or angiotensin determined at 8 weeks contributed to the elevation of blood pressure observed at three weeks. ADRENAL GLANDS Corticosteroid_Synthesis and Metabolism In most mammals, the adrenal glands consist of a cortex of steroid-secreting cells surrounding a medulla containing cells which produce catecholamines. The cortex is further stratified into three histologically distinct layers (Wheater et al., 1979). The zona glomerulosa, in which the cells are arranged in clumps, lies immediately beneath the capsule of the gland. The zona reticularis is adjacent to the medulla, and the cells of this cortical zone are small and arranged in an irregular network. Interposed between the zona glomerulosa and zona reticularis is the broadest layer of the cortex, the zona fasciculata, comprised of columns of cells separated by strands of connective tissue. The blood supply to the adrenals is from three arteries which ramify within the cortex (Wheater et al., 23 1979). Branches of these arteries also descend directly into the medulla and form a network around the chromaffin cells. All of the capillaries and venules of the cortex and medulla drain into the central vein of the medulla; therefore, blood flow in the adrenal is from the outer regions to the inner ones. Three major classes of steroids are synthesized in the adrenal cortex: glucocorticoids, mineralocorticoids, and androgens. The glucocorticoids largely affect intermediary metabolism and have an important role in response of the animal to stress. Mineralocorticoids modulate salt and water homeostasis. The adrenal androgens supplement sex steroids produced by the gonads, and are a major source of androgens in females. The pathways of adrenal cortical steroidogenesis are shown in Figure 3. Cholesterol is the common precursor for all corticosteroids and is stored as esters within lipid drOplets in the cells of the cortex. Although adrenal cells have the capacity to synthesize cholesterol, plasma is the primary source of cholesterol for steroidogenesis (Boyd et al., 1983). The synthesis of pregnenolone involves removal of the side chain of cholesterol following hydroxylation at C and C22 (Sandor et al., 1976; Boyd et al., 21 1983). These reactions are catalyzed by an enzyme complex which contains a hemOprotein, cytochrome P450, and a flavoprotein (NADPH-adrenodoxin reductase). Additionally the non-heme iron-sulfur protein, adrenodoxin, is required (Omura et al., 1966). The availability of cholesterol to the cytochrome P450 which catalyzes the side-chain cleavage (cytochrome P4SOSCC) is the rate-limiting step in corticosteroid synthesis. The transformation of pregnenolone to progesterone occurs on the 24 .muonana mp woumcwamow mum :oHuonvoum cfioowuuooonuocfia was wfioowuuooooan mo mhmznuma onu ma moemucm .xouuoo Hmcouvm osu :« mammcowOVHououm mo mmmSnumm .m omswfim 25 mm1mo 10.0. Hm mm5>xomo>x-m_ no 33588;}. «e mmjioaoina an mmjtamexé u~ mm * Q o.wa«w.ok m.m1¢.~m~ ez.«mm.o m~.hzz.e oeouoeooo n.3«m.MA m.o«m.m- m~.«ox.o mm.«k~.m Honoeoo "no o.z~«m.wm m.n«e.n~z 33.nko.o e~.«om.m oeoaoenoo m.o«z.zo w.e«o.azz oosu~x.o o~.«mo.m Honoeou um